Linking the conformational landscape to enzymatic function through functional site distant mutations

一个正在进行的争论涉及构象运动，通常被称为动力学，在生物分子中所起的作用。 功能。对于酶来说，大规模区域运动的时间尺度恰好类似于 显然是“$$”。 催化率(𝑘！“#这是主要的争议点：DO “$$。在这方面，拟议的研究 构象运动直接影响真实催化速率(𝑘！“#)或𝑘！”# 我将进一步探索构象景观的调制如何在没有 𝑘！“# 影响𝑘！“#和基态结构。这一提议的起源来自我们对人类的工作 鸟苷酸激酶(HGMPK)，一种潜在的治疗癌症甚至SARS-CoV-2的靶点， 这促使我们用核磁共振波谱来解决hGMPK的第一个结构 (PDB：6NUI)。在解决hGMPK结构时，我们表达了一系列七个功能位点距离(FSD)， “$$与荒野相比-- 增强𝑘的hGMPK非同义单核苷酸变体(NsSNV)！“# 类型(Wt)。有趣的是，wt hGMPK及其nsSNV的2D[1H，15N]-HSQC核磁共振谱表明 𝑎𝑝𝑝)用于将GMP绑定到 FSD突变对hGMPK骨架折叠的影响最小，但明显的失败率(𝑘𝑜𝑓𝑓 WT和FSD突变体V91M相差约3000 S-1。我们假设hGMPK的活性可以通过 FSD通过重塑构象景观而发生突变。利用核磁共振波谱和等温 量热法，我们将在以下两个具体目标中检验这一假设。在目标1中，我们将量化影响 从动力学和热力学角度对构象格局上的FSD突变进行研究。结果是 从这一目标出发，将提供关于hGMPK催化和结合方案中哪些部分的全面图景 FSD突变对功能的影响最大。对于目标2，我们将去卷积贡献瞬变 构象景观中的结构通过实验驱动在酶催化中发挥作用 合唱团一代。我们的方案将从无偏分子动力学(MD)中选择hGMPK结构 基于核磁共振测量的剩余偶极耦合和互相关弛豫速率的模拟。这个 集合将有助于识别功能上重要的瞬变构象和评估 FSD突变对主干二面体相关运动的影响。据我们所知，这项建议 提供了第一个示例1。)一种酶的实验驱动的整体生成 生理上相关的时间尺度和2.)用配位体测定热力学和动力学参数 结合到一系列酶变异体上的相同位置。这项建议的影响是直接联系 构象景观与酶功能的关系。这些结果的直接应用包括药物 发现，以构象景观内的结构为目标，而不是基态结构 将带来更好的结果，以及生物分子设计，其中可以实施FSD突变来调整 通过对构象景观的操纵来发挥作用。

An ongoing debate concerns the role conformational motions, often termed dynamics, play in biomolceular funtion. For enzymes, it so happens that the timescales for large-scale domain motions are similar to the apparent "$$). catalytic rate (𝑘!"# This observation is where the major point of contention has developed: do "$$. In this context, the proposed studies conformational motions directly impact the true rate of catalysis (𝑘!"#) or 𝑘!"# will further explore how the modulation of the conformational landscape can indeed fine-tune "$$ without 𝑘!"# impacting 𝑘!"# and the ground state structure. The genesis of this proposal arises from our work with human guanylate kinase (hGMPK), a potential therapeutic target for treating cancer and perhaps even SARS-CoV-2, which motivated us to solve the first structure of hGMPK with nuclear magnetic resonance (NMR) spectroscopy (PDB: 6NUI). While solving the hGMPK structure, we expressed a series of seven functional site distant (FSD), "$$ when compared to the wild- non-synonymous single nucleotide variants (nsSNVs) of hGMPK that enhance 𝑘!"# type (wt). Intriguingly, the 2D [1H,15N]-HSQC NMR spectra of the wt hGMPK and its nsSNVs suggest that the 𝑎𝑝𝑝) for GMP binding to FSD mutations minimally impact hGMPK’s backbone fold, yet the apparent off-rates (𝑘𝑜𝑓𝑓 wt and the FSD mutant V91M differ by ~3000 s-1. We hypothesize that hGMPK’s activity can be modulated with FSD mutants by reshaping the conformational landscape. Utilizing NMR spectroscopy and isothermal calorimetry, we will test this hypothesis in the following two Specific Aims. In Aim 1, we will quantify the impact of the FSD mutations on the conformational landscape from kinetic and thermodynamic perspectives. The results from this Aim will provide a comprehensive picture as to where within the hGMPK catalytic and binding schemes the FSD mutations have the largest impact on function. For Aim 2, we will deconvolute the contribution transient structures within the conformational landscape play in enzymatic catalysis through experimentally driven ensemble generation. Our protocol will select hGMPK structures from unbiased molecular dynamics (MD) simulations based on residual dipolar couplings and cross-correlated relaxation rates measured with NMR. The ensembles will aid in the identification of the functionally important transient conformations and an assessment of the impact the FSD mutations have on backbone dihedral correlated motions. To our knowledge, this proposal provides the first examples 1.) of experimentally driven, ensemble generation for an enzyme spanning physiologically relevant timescales and 2.) of determining thermodynamic and kinetic parameters with ligand binding to the same exact site on a series of enzyme variants. The impact of this proposal is the direct linkage of the conformational landscape to enzymatic function. Immediate applications for these results include drug discovery, where targeting structures within the conformational landscape rather than the ground state structure will lead to better outcomes, and biomolecular design, where FSD mutations can be implemented to adjust function through manipulation of the conformational landscape.